The present invention relates to the field of measuring and analyzing particle distributions. More specifically, the invention relates to a method for analyzing aberrant biological cell populations, particularly hematological populations.
Certain particle size populations that have a typical size distribution are commonly measured and reported by means of a relatively constant mathematical size distribution curve. Among such populations are included red blood cells, bacterial cells, latex particles and other fine particles. Certain characteristics of particle size distributions have been made by comparing sample size distribution data with average, normal size distribution data for the same particles. See, for example, the methods described in U.S. Pat. No. 4,817,446.
Current automated particle size analysis instruments use one or more parameters of light scatter, fluorescence, volume measured by direct current (D.C.), (Coulter) volume, and high frequency (radio frequency), to provide data used to analyze characteristics or properties of particles. Of particular interest are those characteristics or properties of particles that can be used to create a particle distribution curve. For example, some of these instruments are based on the Coulter Principle of detection and measurement of changes in electrical impedance produced when particles (e.g., cells) suspended in a conductive diluent pass through an aperture. Submerged electrodes, through which constant current passes, are located on either side of the aperture. As a dilute suspension of cells is drawn through the aperture, the passage of individual cells momentarily increases the impedance of the electrical path between the two submerged electrodes. See, e.g., FIG. 2. While the number of electrical pulses (i.e., a pulse indicates the time for a specified voltage to pass through the particle and return to a baseline value) indicates cell count, amplitude of the pulses corresponds to cell volume.
A histogram is a graphical representation of the frequency distribution of the cells. By electronically sorting the cells by pulse size (cell volume) and placing each pulse into xe2x80x9cbucketsxe2x80x9d or channels according to the size of the pulse, a histogram can be created. The sorted pulses are displayed as a histogram with volume on the X-axis and pulse frequency (number) on the Y-axis. This process is called channelyzation. The number of channels into which the X-axis is divided, and the size range covered by the channelyzer is fixed in the design of the particular hematology analyzer. These parameters are dependent on the cell population being analyzed and the sensitivity (resolution) required.
Histograms graphically show a cell population""s shape and its spread (i.e., variation around the mean). Thus, histograms provide an assessment of red blood cell (RBC) morphology by the measurement of cell size, Mean Cell Volume (MCV) and Red Cell Distribution Width (RDW). MCV is a measure of the average cell volume in the population (i.e., the population mean). RDW is a measure of the amount of dispersion (or anisocytosis) or heterogeneity in the RBC population. A resulting RBC histogram is typically Gaussian in shape and the distribution of the red cells about the mean is reasonably constant within a population of normal samples. See, for example, FIG. 1.
There is a need in the art for additional methods for determining and identifying particle distributions that fall outside the average histogram mean, or mean of other types of particles. Such particle distributions aid in the diagnosis of disease by differentiating abnormal blood samples from normal blood samples, particularly where the distribution of the particles falling outside of the mean of such xe2x80x9cnormalxe2x80x9d particles is evidence of such abnormality.
In one aspect, the invention provides a method for analyzing an abnormal particle population in an experimental sample containing the particles. The method comprises, as one step, analyzing a particle property distribution curve of the experimental sample in a particle analysis instrument. The instrument provides electrical pulse data of a property of the particle recorded as a range of channel volume numbers and pulse frequency data. A plot of pulse frequency vs. channel number produces a particle property distribution curve. The analysis step is performed to identify particle populations at the lower or upper region of the experimental distribution curve that differ from particle populations in the same regions of an average particle property distribution curve. The average curve is based on multiple normal samples containing the same type of particle. Further, the method includes the step of determining the number or percentage of particles within the lower or upper region of the experimental distribution curve to identify a characteristic of the sample. An increase or decrease in the percentage or number of particles of the experimental sample in these regions of the curve in comparison to the percentage or number in the same regions of the average curve indicates an abnormal particle population in that region. Such an abnormal population of particles is a characteristic of the experimental sample that can be used for diagnosis of disease or evaluation of a product, depending upon the nature of the particles evaluated. In a particularly preferred embodiment, the particle property is particle size.
In one embodiment, the method also provides the steps of determining a particle property distribution index based on the average particle property distribution curve in the particle analysis instrument. The method further involves comparing the average particle property distribution curve and the experimental particle property distribution curve by analyzing curve data using the index. In a particularly preferred embodiment, the particle property is particle size.
In another aspect, the invention provides a method for analyzing an abnormal red blood cell population in an experimental sample comprising the steps of analyzing a red blood cell size distribution curve of the experimental sample in a hematology instrument. This instrument provides electrical pulse size data recorded as a range of channel volume numbers and pulse frequency data. A plot of pulse frequency vs. channel number produces the red blood cell size distribution curve. Red blood cell populations at the lower or upper region of the experimental distribution curve that differ from red blood cell populations in the same regions of an average red blood cell size distribution curve based on multiple normal samples are identified. The number or percentage of red blood cells within the lower or upper region of the experimental distribution curve is determined to identify a characteristic of the sample.
In another aspect, the invention provides an improvement in methods for analyzing particle population in an experimental sample. The method includes the steps of evaluating the sample in a particle size analysis instrument that measures the size and frequency of electrical impedance pulses produced when each particle in a conductive diluent passes through a constant current. This instrument provides pulse size data recorded as a range of channel volume numbers and pulse frequency data. A plot of pulse frequency vs. channel number produces the particle size distribution curve. In such methods, an experimental sample size distribution curve is compared with an average size distribution curve for normal samples of the same particles. The improvement comprises the steps of: (a) identifying differences in the particle populations at the lower or upper region of the experimental sample distribution curve from similarly located particle populations of the average particle size distribution curve; and (b) quantifying the number or percentage of particles within the lower or upper region of the experimental distribution curve that differs from the average curve by an analysis using a particle size distribution index.
In still another aspect, the invention provides a computer program that implements the methods described above. The program implements or performs the following analytical steps in concert with data generated by a particle analysis instrument. An experimental particle property (e.g., size) distribution curve is produced by analyzing an experimental sample containing particles in a particle analysis instrument. The distribution curve of the experimental sample is analyzed to identify particle populations at the lower or upper region of the experimental distribution curve that differ from particle populations in the regions of an average particle property distribution curve based upon normal samples. The program also quantifies the number or percentage of particles within the lower or upper region of the experimental distribution curve. Sorted experimental data is analyzed using a particle property distribution index based on an average particle property distribution curve for multiple normal samples. An increase in the number or percentage of the particles in the lower or upper region identifies a characteristic of the sample. Finally, the program permits the identification of a deviation from the average normal samples in the region by generating an electrical signal.
In yet a further aspect, the invention provides a particle analysis instrument that comprises a computer program as described above. In one aspect, the computer program is integrated into the analysis instrument. In another aspect, the computer program is provided in a separate computer that is a standalone instrument into which is fed data provided by the analysis instrument. In still another aspect, the computer program is associated with the analysis instrument as part of a xe2x80x9cplug-inxe2x80x9d device that can be connected to the instrument.
In still another aspect, the invention provides a parameter for analyzing conditions associated with aberrant red blood cell populations in a histogram. The xe2x80x9cMicroxe2x80x9d parameter is defined as the percentage or number of cells in the region of the histogram spanning the beginning of the experimental sample distribution curve to a threshold related to a particle size distribution index.
In a further aspect, the invention provides another parameter for analyzing conditions associated with aberrant red blood cell populations in a histogram. This xe2x80x9cMacroxe2x80x9d parameter is defined as the percentage of cells in the region of the histogram spanning a threshold related to a particle size distribution index and the end of the experimental sample distribution curve.
Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof.